Phosphorylation of NF2 at Serine-13 by MAP4K family kinases mediates pathological angiogenesis

Cell culture HEK293A cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 50 μg/mL penicillin/streptomycin at 37°C in the incubator containing 5% CO2. MAP4K3-7 5KO, MST1/2 dKO, and MST1/2-MAP4K1-7 9KO cells were described previously (Meng et al, 2015). NF2 KO and MAP4K4 KO cells were generated by CRISPR-Cas9 system. Mouse embryonic fibroblasts (MEF) cells were maintained in DMEM medium containing 10% FBS and 50 μg/mL penicillin/streptomycin. Human umbilical vein endothelial cells (HUVEC) were cultured in Endothelial Cell Growth Medium 2 (PromoCell, C-22011) on plates pre-coated with 1% collagen (Corning, 354236).


2
For gene deletion, CRISPR-Cas9 genomic editing technology was used in this study.
The plasmid px459 v2 was provided by Dr. Feng Zhang (Addgene #62988). The single-guide RNA (sgRNA) sequences targeting individual genes were as follows:

Kinome screening
HEK293A stable cells expressing Ha-tagged NF2 were constructed. Flag-tagged human kinome plasmids (Meng et al., 2015) were transfected into the stable cells individually. Samples were lysed in SDS loading buffer and subjected to immunoblotting. Phospho-specific antibodies for p-NF2 (S13) were used to detect signals.

Kinase assay
Full-length His-tagged human NF2 protein was purified from Escherichia coli . GST-tagged mouse LATS2 protein was expressed in MST1/2-MAP4K1-7 9KO HEK293A cells. After immunoprecipitation from transfected HEK293A cells, MAP4K4 was captured on the beads and incubated with the substrates His-NF2 or GST-LATS2. The kinase reaction was performed for 30 min at 30°C.
Phospho-specific antibodies for p-NF2 (S13) and p-LATS (T1079) were used to evaluate the kinase activity of MAP4K4.

Immunoblotting
Cells were lysed in SDS loading buffer containing 50 mM Tris pH 6.8, 2% SDS, 0.025% bromophenol blue, 10% glycerol, and 5% BME. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. 7.5% phos-tag gel was used to resolve the phospho-NF2 and phospho-YAP proteins. The membrane was blocked in 5% 3 non-fat milk for 1 h at room temperature, and incubated with primary antibodies in 5% bovine serum albumin (BSA) overnight at 4°C. The membrane was then incubated with secondary HRP-conjugated antibodies in 5% non-fat milk for 1 h. Molecular weight marker, ECL substrates, and image acquisition equipment (5200S) were from Tanon Science & Technology Co., Ltd. p-NF2 (S13) rabbit polyclonal antibody was custom-made by Abclonal.

Immunofluorescence
Cells were seeded on fibronectin-coated coverslips and incubated with medium. Cells were then fixed in 4% paraformaldehyde for 15 min, treated with 0.1% Triton X-100 for 10 min, and blocked with 3% goat serum for 1 h. Cells were incubated with primary antibodies overnight at 4°C and Alexa Fluor 488-or Alexa Fluor 594conjugated secondary antibodies for 1 h at room temperature. Slides were mounted with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher, P36935). After mounting, images were captured with Leica SP8 or Zeiss confocal microscope.

RNA extraction, reverse transcription, and real-time PCR
Total RNA was extracted using the RNeasy Plus mini kit (Qiagen). cDNA was generated using the PrimeScript RT reagent Kit (TaKaRa), and quantitative qPCR was conducted using SYBR Green qPCR Master Mix (TaKaRa) on the 7500 real-time 4 PCR system (Applied Biosystems). Relative abundance of mRNA was calculated by normalization to β-actin mRNA. Primers used in PCR were as follows: CYR61,

Isolation of mouse embryonic fibroblasts (MEF)
MEF cells were isolated from mouse embryonic tissue at E11-13. The head above eyes and red tissue (heart and liver) of embryo were cut off. The rest of the embryo were dissected, minced into fragments, and then digested with collagenase/dispase (Roche, 1 mg/mL) in DMEM at 37°C for 30 min and filtered through 40 μm nylon mesh. After centrifugation, the supernatant was removed. MEF cells were resuspended in fresh medium and seeded on the dish.

Isolation of mouse liver endothelial cells
Endothelial cells were isolated from mouse liver between 6 and 8 weeks. The liver was minced, digested with collagenase/dispase (Roche, 1 mg/mL), and dispersed into a single cell suspension. The endothelial cells were purified from cell suspension with CD31 MicroBeads (Miltenyi Biotec, 130-097-418) using a strong magnetic separation method. The purified cells were cultured on dishes pre-coated with 1% collagen in Endothelial Cell Growth Medium 2.

Tube formation assay
HUVECs were used in the tube formation assay. For gene knockdown, siRNA was transfected into HUVECs by Lipofectamine™ RNAiMAX Transfection Reagent (ThermoFisher, 13778075). The sequences of siRNA were as follows: siNF2, GGACAAGAAGGTACTGGATCATGAT. For tube formation assay, HUVECs were resuspended in medium with a concentration of 3x10 5 /mL. Then 200 μL cell 5 suspension were seeded in 48-well plates pre-coated with growth factor reduced Matrigel (Corning, 356231), and underwent angiogenesis in vitro for 8 h. Images were recorded by Olympus microscope. The total length, junctions and meshes of tubes were analyzed by ImageJ.

Whole-mount staining of retinas
The eyes of mouse pups were fixed in 4% paraformaldehyde (Sigma) for 1 h on ice.

Oxygen-induced retinopathy (OIR)
The protocols of OIR were described previously (Smith et al, 1994). In the pathologic mouse model, breeding mother and P7 neonatal pups were exposed to 75% O 2 until P12. Then, pups were exposed to room air for 5 days until P17. Eyes were collected and the retinas were stained with Alexa-Fluor-conjugated isolectin B4. The avascular, sprouting, and tuft areas were measured by ImageJ.

Wound healing assay
In vivo wound healing assay was described previously (Zhang et al, 2016). Mice aged between 6-8 weeks were anesthetized and shaved on the back. Wounds were created using a 6-mm biopsy punch on the back skin. This assay set two wounds per mouse, one on each side of the dorsal midline, at equal distance, so that the tension of skin would be equal all over the mouse back. The images were acquired with digital camera from day 0 to day 8. Wound areas were measured by ImageJ.

Statistical analysis 6
All experiments subjected to statistical test were repeated at least three times.
Statistical analyses were performed using GraphPad Prism8. The independent experimental data were used for Student's t-test. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).  (A) Phosphorylation at S12, S13, and S518 of NF2 as shown by mass spectrometry. were used. Whole cell lysate or precipitated proteins (by Flag antibody) were used to test affinity and specificity of pS13 antibodies.

Supplemental References
(C) Specificity of pS13 antibody. NF2 WT and S13A mutant were transfected into HEK293A cells. pS13 antibody failed to recognize S13A mutant. 8 Figure S2. The effect of NF2 N-terminal residues.
(A) Subcellular localization of WT and mutated NF2. WT or mutated NF2 was transfected into HEK293A cells, and subcellular localization was determined by immunofluorescence staining. Scale bar, 10 μm.
(B) Four serine residue mutants separate on the phos-tag gel. S13A but not other mutants showed compromised phosphorylation. 9 Figure S3. NF2 S13 phosphorylation does not regulate YAP/TAZ activity.
(B) Effects of NF2 WT, S13A, and S13D on the expression of YAP/TAZ target genes.
The mRNA level of CYR61 and CTGF was effectively downregulated in NF2 KO HEK293A cells expressing WT, S13A, or S13D NF2.
(A) Targeting strategy: The first exon was targeted in Nf2 S13A and S13D knockin mouse model, facilitated by CRISPR/Cas9 technology.
(B) Sequencing results of knockin mice.
(C) PCR primers used for genotyping.
There was no significant change between Nf2 WT and mutant MEF cells.
(E) The expression of angiomotin family proteins is not regulated by S13 phosphorylation. The protein levels of AMOT, AMOTL1, and AMOTL2 were not changed in WT, S13A, and S13D MEF cells.